LONP1 targets HMGCS2 to protect mitochondrial function and attenuate chronic kidney disease

Abstract Mitochondria comprise the central metabolic hub of cells and their imbalance plays a pathogenic role in chronic kidney disease (CKD). Here, we studied Lon protease 1 (LONP1), a major mitochondrial protease, as its role in CKD pathogenesis is unclear. LONP1 expression was decreased in human patients and mice with CKD, and tubular‐specific Lonp1 overexpression mitigated renal injury and mitochondrial dysfunction in two different models of CKD, but these outcomes were aggravated by Lonp1 deletion. These results were confirmed in renal tubular epithelial cells in vitro. Mechanistically, LONP1 downregulation caused mitochondrial accumulation of the LONP1 substrate, 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), which disrupted mitochondrial function and further accelerated CKD progression. Finally, computer‐aided virtual screening was performed, which identified a novel LONP1 activator. Pharmacologically, the LONP1 activator attenuated renal fibrosis and mitochondrial dysfunction. Collectively, these results imply that LONP1 is a promising therapeutic target for treating CKD.

1. Figure1: Does downregulation of LONP1 occur at the transcriptional level, or is it due to loss of tubules? Transcriptome analyses, especially at single cell resolution, are needed to confirm these observations. The authors can refer to available datasets, such as E- MTAB-2502(Nature Medicine Vol.21, pages37-46, 2015 and GSE145053 (JASN Vol. 31, Issue 12, pages 2833-2854,2020. To fully assess the impact of LONP1 on renal metabolism, the authors should profile the changes of mitochondrial genes in tubular cells. 2. Figure 2: EM fields of view are notoriously heterogeneous and selective. Please show additional, low magnification images including an intact tubular cell. 3. Figure 3: In 5/6 nephrectomy, fibrosis usually companied with glomerular hyper filtration and sclerosis. Please show a low magnification picture to exclude the edge fibrosis area caused by chopping. Please show low magnification images of tubular mitochondria. Supplemental Figure S3A: the authors mentioned in Method: "injected 2ml of the plasmids to the mice through the tail vein via high-throughput within 10 seconds". At week 8, the mice weighed approximately 25 g and had a blood volume of approximately 5.6% of their body weight (based on the JAX laboratory database), so the total blood volume of the experimental mice was approximately 1.5 ml. Did this protocol cause any problem for mice? 4. Figure 4: the authors showed that LONP1 overexpression in HK2 cells increased the spare respiratory capacity upon TGFb1 treatment. It tends to stand alone as descriptive observations that are not clearly linked mechanistically with suppression of FN, Col, SMA mRNA. Did overexpression of LONP1 also suppress TGFb1 production in vivo? 5. Figure 5: Can the authors provide a list of known substrates of LONP1? How many substrate proteins are present in the proteomics results? Metabolomic profiling or targeted metabolome analysis should be added here to corroborate the results of proteomics. 6. Figure 6: interveinal delivery of DNA plasmid usually fail to archive the desired results; coupled with the problematic "tail vein hyperbaric injection" protocol, this reviewer suggests to perform bioluminescence imaging to verify the distribution of DNA plasmids. 7. Figure 7: What is the half-life and distribution of 84-B10 in the mouse body? In current mouse models of kidney fibrosis, production of matrix proteins by damaged tubular cells is limited. Whereas, TGF-beta1 stimulated the activation/transformation of interstitial fibroblasts contributing largely to matrix production and fibrosis.
Relevant key experiments should be repeated using fibroblasts. 8. Figure 8: did 84-B10 decrease beta-oxidation in tubules of both Sham and UIRI mice? 9. Please proofread the entire article carefully as some sentences are misleading, for example: "To ascertain the role of LONP1 in CKD, we generated proximal tubule Lonp1 conditional knock-in mice (cKI) that had approximately two-fold more proximal tubular cells, compared with those of the WT mice (Supplemental Figure S1A and B). "Firstly, we confirmed EGFP protein expression by fluorescence microscopy were mainly in renal tubules after being overexpressed in the tail vein (Supplemental Figure S3A)".

Referee #2 (Remarks for Author):
In this study, the authors investigated the role of Lon protease 1 (LONP1), a major mitochondrial protease, in CKD. By using human biopsies from patients suffering from CKD, and the UUO and 5/6Nx models of experimental nephropathy, as well as in vitro experiments in tubular cells, they demonstrated that downregulation of LONP1, disrupted mitochondrial function and further promoted CKD progression, whereas its overexpression alleviated the injury. Furthermore, they tested a LONP1 activator, identified by computer virtual screening, that attenuated renal fibrosis, and mitochondrial dysfunction, improving thus CKD progression. Some of the provided pictures are impressive and the paper is very well designed. The manuscript could be strengthened with some additional work.
1-My main concern is the quantification of fibrotic areas using Masson's trichome. This is definitely not appropriate! The authors have to use Sirius Red coloration. I have no doubts about the result as it is clearly demonstrated by the provided WB, that fibrotic markers are increased once LONP1 expression is decreased, and these markers are decreased once LONP1 is overexpressed by tubular cells. 5-The authors used two models of experiment nephropathy, the UUO and 5/6Nx, but to test the 84-B10 activator they used the UUO and the unilateral ischemia-reperfusion injury (UIRI) model of acute injury. What is the reason for not using the 5/6Nx model? By the way, both low and high doses of 84-B10, do not seem to ameliorate renal function in the UIRI model (figS9). Arguments on this point are welcome.
6-Is there a correlation between LONP1 decreased expression and renal function in the patient data? 1

Responses to the Editor:
Physical interaction between 84-B10 and LONP1 should be experimentally shown e.g., by performing Surface Plasmon Resonance.

Response:
We thank the Editor for pointing this out, and have quantified the binding affinity of 84-B10 to recombinant LONP1 protein experimentally by Surface Plasmon Resonance (SPR). LONP1 was diluted to 20 µg/mL and immobilized on a CM5 sensor chip. The binding affinity of 84-B10 (0.3125, 0.625, 1.25, 2.5, 5 and 10 µM in PBS/0.01% Tween 20 buffer) to LONP1 was determined using the Biacore T200 SPR biosensor systems. Result revealed reliable binding activity between 84-B10 and LONP1, with the binding affinities (KD value) calculated as 312.5 nM ( Fig 7E).

Responses to the Referees:
Responses to the Referee #1: Injecting 2ml of plasmid DNA into a 25 g mouse via the tail vein within 10 seconds may lead to ethical concerns.
Response: Thanks for this important comment. Hydrodynamic injection in mice is a method to rapidly inject a large volume of plasmid DNA solution into mice through the tail vein of mice to obtain transgenic expression. It has been reported that when the injection volume accounts for 8%~10% of the body mass of mice, the expression efficacy of transferred genes is the best (PMID: 10455434, Gene Ther. 1999 Jul; 6(7): 1258-66). We and other groups have also used this technique to overexpress genes in

Response (RU)
16th Nov 2022 1st Authors' Response to Reviewers 2 injection, the animal may suffer some bad stress. In our and other groups, this kind of studies was approved by the ethics committees of institutions. Our ethics number is 2007001-7 and 2102005-1, which was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. However, as you mentioned, we cannot completely exclude the potential ethical problems. During the review period, we bred and obtained Hmgcs2 heterozygous knockout mice to perform the UUO model, and found that renal fibrosis and mitochondrial function were significantly improved ( Figure 6H-P). This is consistent with the findings of aggravation of renal fibrosis by overexpressing Hmgcs2 via rapid tail vein injection. Therefore, to avoid the potential ethical concerns, we included the data of knockout mice instead of tail vein injections in our revised MS. Thanks a lot for your important comments for pointing out this potential issue.
In this study, data are presented to suggest a mechanistic pathway involving tubular cell metabolism, transforming growth factor beta 1 (TGF b1) signaling and kidney fibrosis. The results and conclusions support the notion that preservation of renal tubular cell mitochondrial function can block or slow the prognosis of renal fibrosis.
Although there is no striking conceptual advance in this study, it demonstrated, using both genetic and pharmacologic approaches, a new pathway which links LONP1 to HMGCS2 regulating the fibrotic gene expression in tubular cells. Considering that the major contributor to kidney scaring is renal fibroblast, the authors should test the pathway in these cells to support the conclusion. In addition, it would be helpful if more unbiased approaches, (e.g., bulk and single cell transcriptome, metabolomics) were used to support the phenotypes they observed; this would provide a stronger rationale for their experimental proposal.
Response: Thanks very much for your support on our study. Following your valuable comments, we analyzed the Lonp1 mRNA expression in online datasets and our UUO model and found the expression of Lonp1 was both decreased. We performed untargeted metabolomic analyses of UUO models in WT and Lonp1 cKI mice to 3 support our results. We also found that tubular LONP1 silencing promoted fibroblast activation, and 84-B10 inhibited TGF-β1-induced fibroblast activation. The plasma pharmacokinetics (PK) and tissue distribution of 84-B10 in mice were evaluated by

Response:
We sincerely thank the reviewer for the valuable comment. Following your suggestion, we analyzed the expression of Lonp1 in the datasets you mentioned.
Consistent with our results, we found that Lonp1 expression was significantly reduced in CKD patients in the E-MTAB-2502 dataset ( Figure EV1A). The mRNA expression of Lonp1 was also decreased in UUO-7d and R-UUO-4w models in GSE145053 dataset ( Figure EV1B), as well as in our UUO models ( Figure EV1C). Meanwhile, we analyzed the expression of Lonp1 in single-cell sequencing data (Gene Atlas of Reversible Unilateral Ureteric Obstruction Model) and found that Lonp1 was more expressed in the renal tubules and decreased after the UUO model ( Figure EV1D).
According to the analysis of single cell sequencing results, the gene expression is standardized, representing the expression amount of single renal tubular cells.
Therefore, the decrease of LONP1 is not caused by the loss of tubules. Combined with our immunohistochemical results ( Figure 1A), we also found that the expression of LONP1 was reduced in renal tubules and tubular cells. Thus, the expression of tubular Lonp1 was reduced under CKD condition independent of tubular loss.

4
According to your opinion, we detected the expression of mitochondrial genes in the UUO model of Lonp1 cKI mice and found that the expression of almost all mitochondrial genes was decreased in the UUO model, and some genes were up-regulated after Lonp1 overexpression, such as Nd4, Nd4l, Nd6, Co3, Atp6, while some genes were not differentially affected, such as Nd1, Nd2, Nd5, Co1, Co2 and Atp8 ( Figure EV1H and I).  H qRT-PCR analysis of mitochondrial genes (Nd4, Nd4l, Nd6, Co3 and Atp6) in WT and cKI mice after UUO (n=8 in each group, biological replicates).
2. Figure 2: EM fields of view are notoriously heterogeneous and selective. Please show additional, low magnification images including an intact tubular cell.
Response: Thank you very much for the suggestion. In the revised MS, we showed additional low magnification images ( Figure EV2H). We are sorry that we did not photograph the intact renal tubule cells at that time for a clearer view of mitochondria.
But we reproduced electron microscopy in UUO model of Lonp1 cKI mice and photographed intact renal tubule cells and mitochondria ( Figure EV1J). We hope these additional data could address this comment.   Response: Thank you very much for the suggestion. The known substrates of LONP1 are as follows: aconitase (Aco2), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1), steroidogenic acute regulatory protein (Star), succinate dehydrogenase subunit 5 (Sdhaf2), transcription factor A (Tfam) and glutaminase C (Gls), as well as some heme-related enzymes such as cystathionine β-synthase (Cbs), heme oxygenase 1 (Hmox1) and 5-aminolevulinate synthase (Alas1). Most of the substrates were included in the proteomics results, as shown in Figure EV4A, but the differences in expression of these proteins were very small or non-significant between the WT and cKO groups, whereas the Hmgcs2 was significantly different between both groups.  Expression of pyrimidine metabolism-related enzymes in our proteomic data. 11 6. Figure 6: interveinal delivery of DNA plasmid usually fails to archive the desired results; coupled with the problematic "tail vein hyperbaric injection" protocol, this reviewer suggests to perform bioluminescence imaging to verify the distribution of DNA plasmids.
Response: Thanks for the suggestion. During the review period, we bred and obtained Hmgcs2 heterozygous knockout mice to perform the UUO model and the result is consistent with the findings by overexpressing Hmgcs2 via rapid tail vein injection.
Although the method of tail vein injection has been reported in many literatures, in order to avoid the ethical issues, we used Hmgcs2 knockout mice instead of tail vein injection in the revised MS, thus, we did not do bioluminescence experiment due to the deletion of tail vein injection data.

Figure 7: What is the half-life and distribution of 84-B10 in the mouse body?
Response: We appreciate the reviewer's important suggestion, and have evaluated the plasma pharmacokinetics (PK) and tissue distribution of 84-B10 in mice by HPLC coupled with tandem mass spectrometric detection (LC-MS/MS). Detailed methodology was stated in the method part. The plasma-concentration versus time profile (including PK parameters) of 84-B10 was shown in Figure EV5A, and the calculated PK parameters were: T1/2, 2.16 h; Tmax, 0.25 h; Cmax, 4523.0168 nM; AUC0-inf 4653.7194 (h × nmol/L). Tissue concentration of 84-B10 was estimated 30 min after 84-B10 injection (5 mg/kg). As shown in Figure EV5B, 84-B10 was mainly distributed in small intestine (91.34 ± 18.68 nM/g tissue weight), liver (41.68 ± 2.09 nM/g tissue weight) and kidney (24.32 ± 1.72 nM/g tissue weight). In current mouse models of kidney fibrosis, production of matrix proteins by damaged tubular cells is limited. Whereas, TGF-beta1 stimulated the activation/transformation of interstitial fibroblasts contributing largely to matrix production and fibrosis.
Relevant key experiments should be repeated using fibroblasts.

Response:
We agree with the reviewer's suggestions and evaluated the influence of 84-B10 on rat kidney fibroblasts NRK-49F. It is known that injured renal tubules can secrete various factors to act on fibroblasts and promote their activation. Therefore, we interfered LONP1 with shRNA in renal tubular cells for 24 hours and then collected the supernatant to stimulate NRK-49F cells and found that it could promote the activation of fibroblasts (evidenced by increased expression of FN1 and Collagen III) (Appendix Fig S1A-D). We also examined the direct action of 84-B10 on NRK-49F cells. As shown in Appendix Fig S1E- Data information: Data are presented as mean ± SEM. Student's t-test. 9. Please proofread the entire article carefully as some sentences are misleading, for example: "To ascertain the role of LONP1 in CKD, we generated proximal tubule Lonp1 conditional knock-in mice (cKI) that had approximately two-fold more proximal tubular cells, compared with those of the WT mice (Supplemental Figure   S1A and B).
"Firstly, we confirmed EGFP protein expression by fluorescence microscopy were mainly in renal tubules after being overexpressed in the tail vein (Supplemental Figure S3A)".

Response:
We apologize for the unclear description. In the revised MS, we carefully proofread the article. We replaced the sentence "To ascertain the role of LONP1 in CKD, we generated proximal tubule Lonp1 conditional knock-in mice (cKI)…" with "To ascertain the role of LONP1 in CKD, we generated proximal tubule Lonp1 conditional knock-in mice (cKI). Compared with wild-type (WT) mice, the expression of LONP1 in renal tubule cells of cKI mice was approximately doubled (Fig EV1G)".
The other sentence "Next, we generated proximal tubule Lonp1 conditional-knockout (cKO) mice with an approximate 100% reduction in cultured primary proximal tubular cells, compared with those of WT mice" was replaced with "Next, we generated proximal tubule Lonp1 conditional knockout (cKO) mice. Compared with WT mice, LONP1 was almost not expressed in primary proximal tubule cells extracted and cultured from cKO mice". Other references to the tail vein injection have been removed from our revised article

Response to Referee #2:
In this study, the authors investigated the role of Lon protease 1 (LONP1), a major mitochondrial protease, in CKD. By using human biopsies from patients suffering from CKD, and the UUO and 5/6Nx models of experimental nephropathy, as well as 15 in vitro experiments in tubular cells, they demonstrated that downregulation of LONP1, disrupted mitochondrial function and further promoted CKD progression, whereas its overexpression alleviated the injury. Furthermore, they tested a LONP1 activator, identified by computer virtual screening, that attenuated renal fibrosis, and mitochondrial dysfunction, improving thus CKD progression. Some of the provided pictures are impressive and the paper is very well designed. The manuscript could be strengthened with some additional work.
Response: Thank you very much for your support on this research work. We have performed additional experiments and carefully revised the MS according to your valuable suggestions.
1-My main concern is the quantification of fibrotic areas using Masson's trichome. This is definitely not appropriate! The authors have to use Sirius Red coloration. I have no doubts about the result as it is clearly demonstrated by the provided WB, that fibrotic markers are increased once LONP1 expression is decreased, and these markers are decreased once LONP1 is overexpressed by tubular cells.  Response: Thanks for pointing out this phenomenon. The GAPDH antibody we use is a polyclonal antibody, sometimes non-specific bands may appear when the exposure time is long. We are very sorry that we didn't pay attention to this problem previously.
In the revised MS we chose a picture with a shorter exposure time instead as follows. fig 8G there is a problem with the Masson's coloration.

Response:
We thank the reviewer for pointing this out, and have replaced Fig 8G with Sirius Red staining in the revised manuscript. 5-The authors used two models of experiment nephropathy, the UUO and 5/6Nx, but to test the 84-B10 activator they used the UUO and the unilateral ischemia-reperfusion injury (UIRI) model of acute injury. What is the reason for not using the 5/6Nx model?

Response:
We appreciated the reviewer's nice suggestions. 5/6Nx model is a long-term disease model and needs large dose of drugs. Thus, we did not examine the role of 84-B10 in this model previously. In the revised MS, we identified the role of 84-B10 using the 5/6Nx mice model. Consistent with the effect of 84-B10 in UUO and UIRI model, 84-B10 also attenuated renal fibrosis in 5/6Nx mice ( Figure   EV5C-G). The anti-fibrosis effect of 84-B10 in different types of CKD models better indicated its potential in clinic use, as the insults leading to CKD is various in patients. Thus, the increased BUN and Scr levels might reflect a mild re-setup of renal excretion ability in UIRI model because it is known that the healthy kidney in contralateral side is enough to maintain the homeostasis of metabolism. 6-Is there a correlation between LONP1 decreased expression and renal function in the patient data?
Response: Thanks for the suggestion. We analyzed the correlation between LONP1 and BUN and Scr in CKD patients, and found that LONP1 was negatively correlated with BUN and Scr ( Figure EV1E and F). -Please rename "Competing Interest" to "Disclosure Statement & Competing Interests". We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests. Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary. -Author contributions: Please remove it from the manuscript and specify author contributions in our submission system. CRediT has replaced the traditional author contributions section because it offers a systematic machine-readable author contributions format that allows for more effective research assessment. You are encouraged to use the free text boxes beneath each contributing author's name to add specific details on the author's contribution. More information is available in our guide to authors: https://www.embopress.org/page/journal/17574684/authorguide#authorshipguidelines -Please be aware that all deposited datasets should be made freely available upon acceptance, without restriction. Please check "Author Guidelines" for more information. https://www.embopress.org/page/journal/17574684/authorguide#availabilityofpublishedmaterial 4) EV Tables: Pease submit EV tables as separate files and remove them from the main manuscript file. 5) Appendix: Please add page numbers. 6) Synopsis: -Synopsis image: Please resize the visual abstract to 550 px-wide x (250-400)-px high and submit as a high-resolution .jpeg file.
-Synopsis text: I have modified the synopsis text to fit the journal style. Please review it and amend as you see fit by working from the attached document.
-Please check your synopsis text and image before submission with your revised manuscript. Please be aware that in the proof stage minor corrections only are allowed (e.g., typos). 7) For more information: This space should be used to list relevant web links for further consultation by our readers. Could you identify some relevant ones and provide such information as well? Some examples are patient associations, relevant databases, OMIM/proteins/genes links, author's websites, etc... 8) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF. 9) Please provide a point-by-point letter INCLUDING my comments as well as the reviewer's reports and your detailed responses (as Word file).
I look forward to reading a new revised version of your manuscript as soon as possible.

Zeljko Durdevic
Zeljko Durdevic Editor EMBO Molecular Medicine ***** Reviewer's comments ***** Referee #1 (Remarks for Author): This reviewer is satisfied with the professional and rigorous responses provided by the authors. However, there are two minor concerns with the revision: 1) Figure 1J: there are two forms of Tgfb1 can be detected using western blotting, latent and monomer. Authors should state which form is represented in the Figure 1J. 2) Inhibition of Hmgcs2 reportedly promoted fibrosis in liver (Hepatocyte-Macrophage Acetoacetate Shuttle Protects against Tissue Fibrosis, Cell Metabolism, Puchalska et al., 2019, Cell Metabolism 29, 383-398, February 5, 2019. Considering fibrosis is a general reparative or reactive response of organs, this reviewer suggest discussing this disagreement in Discussion section.

12th Dec 2022 2nd Authors' Response to Reviewers
Responses to the Referee #1: 1) Figure 1J: there are two forms of Tgfb1 can be detected using western blotting, latent and monomer. Authors should state which form is represented in the Figure 1J.
Response: Thanks for this important comment. What we showed in Figure 1J is the monomer form of TGF-β1 (15kD), and we annotated it in the figure legends. Considering fibrosis is a general reparative or reactive response of organs, this reviewer suggest discussing this disagreement in Discussion section.

Response:
We sincerely thank the reviewer for the valuable comment. We have discussed this disagreement in Discussion section: "It has been reported that the inhibition of HMGCS2 in the liver will cause the impairment of hepatic ketogenesis, We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.